Two PD-L1 antibodies (clone name SP263 or 22C3) were used to detect PD-L1 expression. Sp263 was a companion diagnostic assay for OPDIVO® (nivolumab), and 22c3 was a companion diagnostic assay for KEYTRUDA® (pembrolizumab). We performed SP263 and/or 22C3 assays prior to PD-1 inhibitor treatment for all NSCLC patients. Thirteen (25%) of the 52 specimens were tested for both SP263 and 22C3, 27 (51.9%) for only SP263, and 12 (23.1%) for only 22C3. Two PD-L1 tests used prediluted antibody (ready to use) according to the protocol. The SP263 assay was performed using a VENTANA BenchMark ULTRA instrument (Roche, Basel, Switzerland), and the 22C3 assay was conducted using the Dako Link-48 platform (Dako, Carpinteria, California, US), as recommended by the manufacturers [16 (link)]. PD-L1 intensity was also evaluated on a four-point intensity scale (0, none; 1, faint; 2, moderate; and 3, strong), and the percentage of membranous expression of PD-L1 was determined (Figures S2 and S3 ). When both the 22C3 and SP263 tests were conducted, mean values were used. High PD-L1 expression was defined as ≥ 50% of definitive tumor cells exhibiting PD-L1 staining, because 50% was the cut-off used for NSCLC [17 (link)].
Dako link 48 platform
Manufactured by Agilent Technologies
Sourced in United States
The Dako Link 48 platform is a modular and automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining system designed for efficient and consistent sample processing in clinical and research laboratories. It offers automated slide processing capabilities to streamline workflow and facilitate reproducible results.
Automatically generated - may contain errors
4 protocols using dako link 48 platform
1
Evaluation of PD-L1 Expression in NSCLC
2
PD-L1 Expression in Tumor Tissues
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Tumor tissues were obtained from biopsy or surgery, fixed, embedded, and then cut
into 5-µm sections and stained for PD-L1 (clone 22C3, Dako, Agilent, CA, USA),
using the Dako Link 48 platform (Dako). The staining was evaluated by two
independent pathologists in a blinded fashion. Positive expression was defined
as expression in >1% of the tumor.
into 5-µm sections and stained for PD-L1 (clone 22C3, Dako, Agilent, CA, USA),
using the Dako Link 48 platform (Dako). The staining was evaluated by two
independent pathologists in a blinded fashion. Positive expression was defined
as expression in >1% of the tumor.
3
PD-L1 Expression Immunohistochemistry
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Representative tumor sections were immunohistochemically stained for PD‐L1 expression using the standardized 22C3 pharmDx assay on the Dako Link 48 platform (Dako). This assay was used as standard in the KEYNOTE‐048.2
4
Quantifying Tumor Immune Infiltrates
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PD-L1 expression of the tumor samples was measured using the FDA-cleared 22C3 assay on the Dako Link 48 platform (DAKO, clone number 22C3, 1:50 dilution,). PD-L1 expression was reported as combined positive score (CPS), defined as the number of PD-L1-positive cells (tumor cells, lymphocytes, and macrophages) divided by the total number of tumor cells multiplied by 100. Stromal tumor-infiltrating lymphocytes (TILs) were evaluated in hematoxylin and eosin sections following criteria proposed by the International Immuno-Oncology Biomarker Working Group39 (link). Staining of CD8, PD-L1 and TLS were performed on FFPE sections. For immunophenotypes analysis, CD8 was stained with immunofluorescence using primary rabbit anti-human CD8 antibody (Thermo Fisher, catalog number MA5-14548, 1:200 dilution). TLS was stained by multiplex immunohistochemistry. Antibodies used for TLS analysis were primary rabbit anti-human CD4 antibody, (Abcam, catalog number ab133616, 1:500 dilution), primary rabbit anti-human CD8 antibody, (Abcam, catalog number ab93278, 1:4000 dilution), and primary rabbit anti-human CD20 antibody, (Abcam, catalog number ab78237, 1:50 dilution). CD20, CD8, and CD4 enriched area were defined as TLS.
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